The project goal is to understand the selective pressures associated with Wolbachia-mosquito-Chikungunya virus (CHIKV) co-adaptation. New approaches to preventing CHIKV are needed because the endemic range of this virus is expanding and because current methods are limited to controlling mosquito populations, and they cannot prevent invasion of this virus into new locales. A promising candidate for arbovirus control and prevention relies on the introduction of the intracellular bacterium Wolbachia into Ae. aegypti mosquitoes. Wolbachia biocontrol has advanced from laboratory experiments demonstrating that Wolbachia shortens the lifespan of the mosquito while simultaneously reducing virus replication to small-scale field trials demonstrating that Wolbachia are capable of spreading through wild Ae. aegypti populations. This primarily has been proposed as a tool to control dengue virus (DENV) transmission; however, Wolbachia infections confer protection for their insect hosts against a range of pathogens including for Ae. aegypti against CHIKV. A critical next step is to assess the impact of Wolbachia on virus evolution to determine whether or not Wolbachia biocontrol is a sustainable method for CHIKV control and prevention that will not be undermined by mosquito or virus evolution. Accordingly, through this NIH/NIAD R21 we will perform experimental evolution studies to evaluate and anticipate evolutionary changes of CHIKV-Wolbachia-mosquito interactions. Furthermore, Wolbachia-infected Ae. aegypti colonies have been established by a complementary Eliminate Dengue Program (EDP) project in Colombia to evaluate the impact of Wolbachia biocontrol on DENV transmission. There are two specific aims 1.) Evaluate the specific CHIKV phenotypes that arise during alternate passage between Wolbachia-infected Ae. aegypti and mice and 2.) Identify the genetic correlates of adaptation identified in Aim 1 and evaluate their role and importance in facilitating evolution in Wolbachia-infected Ae. aegypti. Experimental evolution of a CHIKV via alternate passage between Wolbachia-infected mosquitoes and mice is new. It is expected that medium-scale Wolbachia deployments will occur in the very near future. Therefore, assessing the evolutionary potential of CHIKV to adapt to Wolbachia-infected Ae. aegypti will help inform the viability of Wolbachia biocontrol for CHIKV control. Potentially, through this R21, valuable evidence could be provided that justifies expanding this type of control program to other Ae. aegypti-transmitted arboviruses, e.g., CHIKV.